Functional analysis of HvSPY, a negative regulator of GA response, in barley aleurone cells and Arabidopsis

SPINDLY (SPY) is an important regulator of plant development, and consists of an N-half tetratricopeptide repeat (TPR) domain containing 10 TPR motifs and a C-half catalytic domain, similar to O-GlcNAc transferase (OGT) of animals. The best characterised role of SPY is a negative regulator of GA signalling, and all known spy alleles have been isolated based on increased GA response. Of the eight alleles that directly affect the TPR domain, all alter TPRs 6, 8 and/or 9. To test the hypothesis that a subset of TPRs, including 6, 8 and 9, are both essential and sufficient for the regulation of GA response, we overexpressed the full-length barley (Hordeum vulgare L.) SPY protein (HvSPY) and several deletion mutants in barley aleurone cells and in Arabidopsis wild type (WT) and spy-4 plants. Transient assays in barley aleurone cells, that also express endogenous HvSPY, demonstrated that introduced HvSPY and HvTPR inhibited GA(3)-induced alpha-amylase expression. With the exception of HvSPY Delta 1-5, the other deletion proteins were partially active in the barley assay, including HvSPY Delta 6-9 which lacks TPRs 6, 8 and 9. In Arabidopsis, analysis of seed germination under a range of conditions revealed that 35S:HvSPY increased seed dormancy. Hvspy-2, which lacks parts of the eighth and ninth TPRs, was able to partially complement all aspects of the spy-4 phenotype. In the presence of AtSPY, 35S:HvTPR caused some phenotypes consistent with a decrease in GA signalling, including increased seed sensitivity to paclobutrazol and delayed flowering. These plants also possessed distorted leaf morphology and altered epidermal cell shape. Thus, despite genetic analysis demonstrating that TPRs 6, 8 and 9 are required for regulation of GA signalling, our results suggest that these TPRs are neither absolutely essential nor sufficient for SPY activity.